The objective of this research proposal is to study the expression and regulation of the puromycin-sensitive aminopeptidase (enkephalin- degrading aminopeptidase), which has been shown to degrade neuropeptides including the enkephalins and dynorphins. Recently, three isoforms of this enzyme were isolated from a cDNA library. We will utilize these cDNAs to isolate a genomic clone(s) of the enzyme to determine the origin of these isoforms. It is expected that the three isoforms arise from a single gene due to the near identity of the majority of their coding sequences. Exons of the genomic clone will be sequenced to determine their splicing pattern. We will determine how the enzyme is regulated in cells, including where each isoform is located in the cell, whether or not each isoform is catalytically active, and how these isoforms are expressed spatially and temporally. Finally, transgenic mice will be employed to study the putative role of the enzyme isoforms on development and on regulation of the cell cycle.